Tuberculin Skin Test (TST) was developed by Robert Koch in 1890 and still is the method of choice in the diagnosis of Tuberculosis (TB). TST measures an in vivo cellular reaction mediated by Th1 cells at the site of tuberculin injection. Although TST detects previous exposure to Mycobacterium tuberculosis (Mtb), it presents low specificity and is not capable to differentiate latent infection from active TB disease. Few years ago, the identification of highly specific RD1 region in Mtb genome drove the development of new in vitro TB specific tests. The commercially available QuantiFERON(r) TB Gold IN TUBE6 is an example of such in vitro tests (also known as IGRA). IGRA detects IFN-? released from lymphocytes after in vitro incubation with Mtb antigens. As opposed to TST, IGRA offer enhanced specificity and are not influenced by cross-reactivity to BCG and other non-tuberculous mycobacteria (NTM). However, it is still controversial whether IGRA can replace TST as the diagnostic test of choice. Although IGRA is, by definition, more specific than TST, several studies have reported an elevated frequency of IGRA reversions and conversions7-15 as well as discordance between IGRA and TST 4, 16, 17. We, recently, reported a high discordance rate (21%) between the two tests, with the majority of discordance coming from HHC with TST positive and IGRA negative results. These findings18 allow us to differentiate Mtb-exposed individuals from uninfected (TST negative, IGRA negative), new infection (TST positive/converted, IGRA negative) and remote infection (TST and IGRA positive). Using samples from these three groups, we propose to dissect the mechanism for TST/IGRA discordancy and to identify biomarkers predictive of new infection. We posit that in the in-vivo TST, recruitment of antigen-specific T cells and T regulatory cells (Tregs) to the injection site i temporally segregated. Whereas in the in-vitro IGRA concurrent presence of antigen-specific T cells and Tregs attenuates antigen-specific T cell expansion and attendant IFN- secretion, leading to differences in sensitivity and a delayed IGRA conversion. Our finding of TST and IGRA discordant subjects is a significant aspect of our proposal since without this group we would have missed identification of biomarkers of new infection. Comparative gene expression analysis of stimulated whole blood cells from the three groups of subjects will allow the identification of genes that are only expressed in newly and recently-infected contacts, with the potential to provide biomarker(s) that can distinguish these two groups of LTBI with the highest risk of progression to disease from the larger group of LTBI with remote infection. The knowledge gained with this proposal will help with the elucidation of immunological basis of TST/IGRA discordance, which in turn may allow the improvement of in-vitro TB diagnostics. Another significant contribution is that the biomarkers of new infection will permit distinction between new and remote infection, allowing identification of newly infected individuals for prophylaxis.